Kristan Melford

Chylomicronemia With a Mutant GPIHBP1 (Q115P) That Cannot Bind Lipoprotein Lipase

Objective—GPIHBP1 is an endothelial cell protein that binds lipoprotein lipase (LPL) and chylomicrons. Because GPIHBP1 deficiency causes chylomicronemia in mice, we sought to determine whether some cases of chylomicronemia in humans could be attributable to defective GPIHBP1 proteins. Methods and Results—Patients with severe hypertriglyceridemia (n=60, with plasma triglycerides above the 95th percentile for age and gender) were screened for mutations in GPIHBP1. A homozygous GPIHBP1 mutation (c.344A>C) that changed a highly conserved glutamine at residue 115 to a proline (p.Q115P) was identified in a 33-year-old male with lifelong chylomicronemia. The patient had failure-to-thrive as a child but had no history of pancreatitis. He had no mutations in LPL, APOA5, or APOC2. The Q115P substitution did not affect the ability of GPIHBP1 to reach the cell surface. However, unlike wild-type GPIHBP1, GPIHBP1-Q115P lacked the ability to bind LPL or chylomicrons (d < 1.006 g/mL lipoproteins from Gpihbp1−/− mice). Mouse GPIHBP1 with the corresponding mutation (Q114P) also could not bind LPL. Conclusions—A homozygous missense mutation in GPIHBP1 (Q115P) was identified in a patient with chylomicronemia. The mutation eliminated the ability of GPIHBP1 to bind LPL and chylomicrons, strongly suggesting that it caused the patient’s chylomicronemia.

Abnormal Patterns of Lipoprotein Lipase Release into the Plasma in GPIHBP1-deficient Mice

GPIHBP1-deficient mice (Gpihbp1–/–) exhibit severe chylomicronemia. GPIHBP1 is located within capillaries of muscle and adipose tissue, and expression of GPIHBP1 in Chinese hamster ovary cells confers upon those cells the ability to bind lipoprotein lipase (LPL). However, there has been absolutely no evidence that GPIHBP1 actually interacts with LPL in vivo. Heparin is known to release LPL from its in vivo binding sites, allowing it to enter the plasma. After an injection of heparin, we reasoned that LPL bound to GPIHBP1 in capillaries would be released very quickly, and we hypothesized that the kinetics of LPL entry into the plasma would differ in Gpihbp1–/– and control mice. Indeed, plasma LPL levels peaked very rapidly (within 1 min) after heparin in control mice. In contrast, plasma LPL levels in Gpihbp1–/– mice were much lower 1 min after heparin and increased slowly over 15 min. In keeping with that result, plasma triglycerides fell sharply within 10 min after heparin in wild-type mice, but were negligibly altered in the first 15 min after heparin in Gpihbp1–/– mice. Also, an injection of Intralipid released LPL into the plasma of wild-type mice but was ineffective in releasing LPL in Gpihbp1–/– mice. The observed differences in LPL release cannot be ascribed to different tissue stores of LPL, as LPL mass levels in tissues were similar in Gpihbp1–/– and control mice. The differences in LPL release after intravenous heparin and Intralipid strongly suggest that GPIHBP1 represents an important binding site for LPL in vivo.

Heparin-binding defective lipoprotein lipase is unstable and causes abnormalities in lipid delivery to tissues

Lipoprotein lipase (LpL) binding to heparan sulfate proteoglycans (HSPGs) is hypothesized to stabilize the enzyme, localize LpL in specific capillary beds, and route lipoprotein lipids to the underlying tissues. To test these hypotheses in vivo, we created mice expressing a human LpL minigene (hLpL(HBM)) carrying a mutated heparin-binding site. Three basic amino acids in the carboxyl terminal region of LpL were mutated, yielding an active enzyme with reduced heparin binding. Mice expressing hLpL(HBM) accumulated inactive human LpL (hLpL) protein in preheparin blood. hLpL(HBM) rapidly lost activity during a 37 degrees C incubation, confirming a requirement for heparin binding to stabilize LPL: Nevertheless, expression of hLpL(HBM) prevented the neonatal demise of LpL knockout mice. On the LpL-deficient background hLpL(HBM) expression led to defective targeting of lipids to tissues. Compared with mice expressing native hLpL in the muscle, hLpL(HBM) transgenic mice had increased postprandial FFAs, decreased lipid uptake in muscle tissue, and increased lipid uptake in kidneys. Thus, heparin association is required for LpL stability and normal physiologic functions. These experiments confirm in vivo that association with HSPGs can provide a means to maintain proteins in their stable conformations and to anchor them at sites where their activity is required.

Heparin decreases the degradation rate of hepatic lipase in Fu5AH rat hepatoma cells. A model for hepatic lipase efflux from hepatocytes

The mechanism for the stimulation of hepatic lipase secretion by heparin was studied in cultured Fu5AH rat hepatoma cells. Quantitative immunoprecipitation followed by electrophoresis and fluorography were used to isolate and quantitate the radioactive enzyme; hepatic lipase protein mass was quantitated by ELISA. Addition of heparin to the medium resulted in a 2-fold increase in lipase secretion rate, whereas cell-surface-associated and intracellular lipase decreased by 76 and 20%, respectively. Rates of synthesis of hepatic lipase measured by incorporation of Trans 35S-label into enzyme protein were not different in control or heparin-treated dishes. In pulse-chase studies, it was estimated that the degradation rate constants for control and heparin-treated cultures were 0.51 +/- 0.09 and 0.14 +/- 0.13 h-1 for control and heparin-treated cultures, respectively. 52% of the synthesized enzyme was degraded in control cultures; addition of heparin to the culture medium reduced this figure to 11% of the synthetic rate. Equilibrium binding data of highly purified 125I-hepatic lipase to Fu5AH cells at 4 degrees C demonstrate the presence of a class of high-affinity binding sites. At 37 degrees C, cell-surface-bound 125I-hepatic lipase is internalized and either degraded or recycled to the medium. The half-intracellular residence times of hepatic lipase were 55 and 31 min in control and heparin-treated cultures, respectively. Radioactivity incorporated in the 55.4 kDa high-mannose-containing lipase and the mature 57.6 kDa species was measured as a means of locating the enzyme in the secretory pathway before or beyond the medial Golgi. The disappearance of the 55.4 kDa species from the cell is similar in control and heparin-treated cultures with half-intracellular residence times of 29 and 25 min, respectively. In contrast, the amount of radiolabeled 57.6 kDa species in control cells remained constant from 15 min to 2 h, whereas it decreased by 79% in heparin-treated cells. The above data demonstrate that the increase in hepatic lipase secretion is due to a decreased degradation rate with no change in synthetic rate and that heparin primarily affected the residence time of hepatic lipase in the medial Golgi-plasma membrane region.

Interaction of lipoprotein lipase and receptor-associated protein.

Receptor-associated protein (RAP) is a recognized chaperone/escort protein for members of the low density lipoprotein receptor family. In this report, we show that RAP binds to lipoprotein lipase (LPL) and may play a role in the maturation of LPL. Binding of highly purified RAP to LPL was demonstrated in vitro by solid phase assays, surface plasmon resonance, and rate zonal centrifugation. The dissociation constant for this interaction measured by the first two techniques ranged between 2.4 and 13 nm, values similar to those reported for the binding of RAP to LRP or gp330. The specificity of the interaction was demonstrated by competition with a panel of LPL monoclonal antibodies. Rate zonal centrifugation demonstrated the presence of a stable complex with an apparent Mr consistent with the formation of a complex between monomeric LPL and RAP. RAP·LPL complexes were co-immunoprecipitated in adipocyte lysates or from solutions of purified LPL and RAP. The interaction was also demonstrated in whole cells by cross-linking experiments. RAP-deficient adipocytes secreted LPL with a specific activity 2.5-fold lower than the lipase secreted by control cells. Heparin addition to cultured RAP-deficient adipocytes failed to stimulate LPL secretion in the medium, suggesting defective binding of the lipase to the plasma membrane. These studies demonstrate that RAP binds to LPL with high affinity both in purified systems and cell extracts and that RAP-deficient adipocytes secrete poorly assembled LPL. A function of RAP may be to prevent premature interaction of LPL with binding partners in the secretory pathway, namely LRP and heparan sulfate proteoglycan.

In Vivo Arterial Lipoprotein Lipase Expression Augments Inflammatory Responses and Impairs Vascular Dilatation

Objective—Although epidemiologic data suggest that hypertriglyceridemia and elevated plasma levels of fatty acids are toxic to arteries, in vitro correlates have been inconsistent. To investigate whether increased endothelial cell expression of lipoprotein lipase (LpL), the primary enzyme creating free fatty acids from circulating triglycerides (TG), affects vascular function, we created transgenic mice that express human LpL (hLpL) driven by the promoter and enhancer of the Tie2 receptor. Methods and Results—Mice expressing this transgene, denoted EC-hLpL and L for low and H for high expression, had decreased plasma TG levels compared with wild-type mice (WT): 106±31 in WT, 37±17 (line H), and 63±31 mg/dL (line L) because of a reduction in VLDL TG; plasma cholesterol and HDL levels were unaltered. Crossing a high expressing EC-hLpL transgene onto the LpL knockout background allowed for survival of the pups; TG in these mice was approximately equal to that of heterozygous LpL knockout mice. Surprisingly, under control conditions the EC-hLpL transgene did not alter arterial function or endothelial cell gene expression; however, after tumor necrosis factor (TNF)-&agr; treatment, arterial vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and endogenous TNF-&agr; mRNA levels were increased and arteries had impaired endothelium-dependent vasodilatation. This was associated with reduced eNOS dimers. Conclusions—Therefore, we hypothesize that excess vascular wall LpL augments vascular dysfunction in the setting of inflammation.

Mice Expressing Only Covalent Dimeric Heparin Binding-deficient Lipoprotein Lipase MUSCLES INEFFICIENTLY SECRETE DIMERIC ENZYME

Lipoprotein lipase (LpL) hydrolyzes triglycerides of circulating lipoproteins while bound as homodimers to endothelial cell surface heparan sulfate proteoglycans. This primarily occurs in the capillary beds of muscle and adipose tissue. By creating a mouse line that expresses covalent dimers of heparin-binding deficient LpL (hLpLHBM-Dimer) in muscle, we confirmed in vivo that linking two LpL monomers in a head to tail configuration creates a functional LpL. The hLpLHBM-Dimer transgene produced abundant activity and protein in muscle, and the LpL was the expected size of a dimer (∼110 kDa). Unlike the heparin-binding mutant monomer, hLpLHBM-Dimer had the same stability as nonmutated LpL. The hLpLHBM-Dimer transgene prevented the neonatal demise of LpL knockout mice; however, these mice were hypertriglyceridemic. Postheparin plasma LpL activity was lower than expected with the robust expression in muscle and was no longer covalently linked. Studies in transfected cells showed that Chinese hamster lung cells, but not COS cells, also degraded tandem repeated LpL into monomers. Thus, although muscle can synthesize tethered, dimeric LpL, efficient production of this enzyme leading to secretion, and physiological function appears to favor secretion of a noncovalent dimer composed of monomeric subunits.